EP2553465A1 - Method to detect tissue degradation leading to inflammation - Google Patents
Method to detect tissue degradation leading to inflammationInfo
- Publication number
- EP2553465A1 EP2553465A1 EP11763156A EP11763156A EP2553465A1 EP 2553465 A1 EP2553465 A1 EP 2553465A1 EP 11763156 A EP11763156 A EP 11763156A EP 11763156 A EP11763156 A EP 11763156A EP 2553465 A1 EP2553465 A1 EP 2553465A1
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- complex
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
Definitions
- the present invention relates generally to methods and products for determining a tissue degradation process and particularly events related to inflammation and propagation in joint disease.
- This invention relates to an assay that can be used to analyze serum, and other human samples (including but not limited to synovial fluid), for the presence of a COMP fragment complex that have activated the complement system exemplified by the complex between COMP and complement factor C3b or natural breakdown fragments of C3b.
- RA rheumatoid arthritis
- OA osteoarthritis
- Tissue degradation processes are a result of the breakdown of tissue molecular constituents. This can be triggered by e.g.
- tissue degradation processes for the purpose of diagnosis, disease monitoring, treatment etc. can be performed by numerous methods.
- One way to determine degradation processes in connective tissue diseases, such as arthritic conditions; artheriosclerosis, degenerative joint conditions etc. is the detection of the presence of degradation products of the connective tissue components. This allows direct detection of the degradation process, compared to indirect methods as e.g. measuring increased amounts of leukocytes, which have been employed in the diagnosis of inflammatory processes such as arthritic conditions.
- Another parameter is the late in the process observed tissue loss detected by imaging methods such as X-ray or MRI.
- Cartilage Oligomeric Matrix Protein (COMP or Thrombospondin V). Elevated serum level of COMP has previously been associated with joint destruction in RA.
- COMP is a structural component of cartilage where it in the growing tissue appears to serve as a catalyst of collagen fibrillogenesis and in the older individual appears to have a structural role in maintaining tissue integrity. Further COMP is a pentameric glycoprotein with a predominant expression in cartilage. This protein is one of the main molecular markers for joint destruction, and elevated levels of COMP can be found both in the synovial fluid and in the serum of patients with active joint disease [1 , 2]. COMP is found most abundantly in cartilage [3] and pressure loaded parts of tendon. In addition, some expression has been reported in synovial and dermal fibroblasts as well as in the blood vessel wall in
- COMP has also been proposed to mediate attachment of chondrocytes to the extracellular matrix through interactions with cell-surface integrins.
- Structurally COMP is a pentamer consisting of five identical subunits that are linked together by a coiled coil structure close to the N-terminus.
- the N-terminus is followed by four epidermal growth factor (EGF) domains, eight thrombospondin type 3 (TSP3) repeats and a globular C-terminus [5]. Mutations in COMP have been shown to lead to pseudoachondroplasia and multiple epiphyseal dysplasia.
- EGF epidermal growth factor
- TSP3 thrombospondin type 3
- the complement system is an array of factors that can rapidly be activated in defense as a part of the innate immune system. Complement activates inflammatory responses and recruits immune cells to the site. It also assists the adaptive immune system in e.g. clearing of damaged and dying cells, misfolded proteins, pathogens and foreign matters from an organism. Yet another important event is opsonisation of the target recognized by
- the complement system consists of a number of proteins normally circulating as inactive precursors (pro-proteins). Over 30 proteins and protein fragments make up the complement system, including glycosylated serum proteins and cell membrane receptors. These proteins are synthesized mainly in the liver, and they account for about 5% of the globulin fraction of blood serum. When stimulated by one of several triggers, proteases in the system cleave specific proteins to release chemoattractant anaphylatoxins and initiate an amplifying cascade of further cleavages. The end-result of this activation cascade is massive amplification of the response with formation of factors active in cell recruitment and with final activation of the pore-forming membrane attack complex.
- the complement system is a sensor of danger aiding in the removal of apoptotic and necrotic cells and immune-complexes as well as a defense mechanism against foreign pathogens.
- Uncontrolled complement activation can on the other hand contribute to several autoimmune disorders and pathological inflammatory conditions. Activation of complement has been shown to occur in the joints of patients suffering from RA and the involvement of complement in pathology of RA has been corroborated in animals models [6].
- the classical complement pathway is typically triggered by immune complexes, whereas the lectin pathway is initiated by specific carbohydrate structures present on pathogenic surfaces.
- the alternative pathway is an auto activation pathway that also serves as an amplification loop for the other two pathways. Recently, it has been confirmed that properdin can directly activate the alternative pathway [7]. In all three pathways of complement activation the crucial step is the proteolytic conversion of the component C3 to C3b.
- Cleavage of C3b by enzymes of the complement cascades will allow the covalent attachment of C3b onto antigen surfaces via the thioester bond capturing either amino or hydroxyl group. This is the initial step in complement opsonisation, and subsequent proteolysis of the bound C3b by complement inhibitor factor I produce iC3b, C3c and C3dg, fragments that are recognized by different receptors.
- C3 is a complex and flexible protein consisting of 13 distinct domains.
- Complement can also be triggered by a number of endogenous ligands, such as members of the small leucine-rich repeat protein (SLRP) family [8].
- SLRP small leucine-rich repeat protein
- the SLRPs have several roles in cartilage, e.g. in contributing to the structural stability of the tissue. During pathogenic cartilage destruction, SLRPs are fragmented and released into the synovial fluid where they can interact with complement. This has been proposed to contribute to the local inflammatory milieu in joints of patients suffering from joint diseases such as RA and OA, where inflammation is prevalent.
- the present invention uses a novel approach to identify the specific process of complement
- WO0138876 there is an assay described for a method of analyzing the presence of human COMP in a clinical sample using a sandwich-ELISA technology with two monoclonal antibodies produced from cell lines.
- the methods described in WO0138876 can partly be used in the present invention, which is hereby fully incorporated by reference, however there is no information therein on the concept now presented regarding a role of COMP in complement activation or complex formation with C3b.
- WO05116658 there is an assay described for determining a tissue degradation process by detection of COMP neoepitopes from the same group as the present invention. Parts of the methods described in WO05116658 can be used in the present invention, which is hereby fully incorporated by reference, however there is no information therein regarding also assaying complexes between COMP and another factor such as C3b.
- US2008233113 relates to a method of inhibiting complement activation by C3b inhibitors in a subject but it does not relate to an assay for an agent activating complement and the resulting complexes between COMP and C3b. Neither is the procedure applied to showing that there is complement activation in the synovial fluid of both OA and RA relating to the COMP release.
- WO2004031240 describes a method to inhibit inflammatory reactions in vivo, more specifically the activation of the complement system.
- the invention consists of the
- WO2008154251 the invention concerns specific antibodies to C3b and the prevention and treatment of complement-associated disorders using such antibodies.
- the invention does not relate to an assay of complexes between COMP and C3b.
- COMP -release from cartilage has been shown to be an early event in both RA and OA preceding radio logically observed cartilage damage. Elevated levels of COMP can therefore be detected both in the synovial fluid and in the serum of patients with active disease.
- Complement activation has been suggested to be one of the factors sustaining the inflammatory state in the joint, especially since the discovery of the activation of the classical pathway of complement by certain cartilage proteoglycans.
- Complement activation products of both the classical and alternative pathways can be found in synovial fluids of patients with active RA, a scenario supported by several animal models showing the protective effect of deficiencies of complement proteins in arthritis disease models as well as therapeutic effects of complement inhibition [6].
- COMP-C3b complexes are present in both serum and synovial fluid of RA patients as an indication of COMP-induced complement activation in vivo.
- the COMP-levels in synovial fluid of RA patients are significantly higher than in serum whereas the COMP-C3b levels are somewhat lower compared to serum.
- One theory is that the events in blood reflects many joints which may contribute differently and another is that complement as well as total protein levels are in general much lower in synovial fluid that in serum and the availability of properdin and C3 might be a restricting factor.
- the invention herein is based on the findings of the role of COMP in complement regulation and that COMP is able to induce complement activation through the alternative pathway.
- COMP-C3b complexes By detecting COMP-C3b complexes in serum and not only COMP it is possible to identify active disease, for example RA or OA, in patients where circulating levels of COMP may remain in the normal range. These complexes are included in the method, in the assay and the kit according to the present invention.
- the invention will also prove useful in identifying the role of complement activation driven by fragments released from the cartilage in other inflammatory conditions with COMP release, e.g. spondylarthritides and juvenile idiopathic arthritis, as well as other conditions like systemic sclerosis and tendon disease.
- This will provide an additional valuable tool for quickly establishing a diagnosis, select and start an early treatment of patients to avoid extensive joint destruction.
- the finding and the invention e.g. a bioassay detecting covalent complexes between COMP and complement component C3b, or natural breakdown products of C3b, will detect a new biomarker for RA disease specifically useful for monitoring disease treatment.
- the assay will also likely identify a subgroup of RA patients particularly amenable to treatment with emerging complement inhibitors.
- the current invention shows a new method to detect ongoing complement activation induced by the tissue destroying process.
- COMP-C3b complexes in serum, and not only COMP it is possible to obtain a several fold more sensitive and specific detection of RA.
- identifying such complexes in the synovial fluid of patients with joint disease it is possible to identify a local activation of inflammation resulting from the cartilage breakdown.
- cardiovascular disease including atherosclerosis
- cardiovascular disease affecting major blood vessels
- cardiovascular disease can be analyzed for contents of COMP-C3b complexes.
- a primary object of the present invention is to provide an assay that can be used to measure and to monitor the success of ongoing treatment regimes, i.e. the signal in the COMP-C3b assay follows disease activity in the patient.
- Another object of the present invention is to provide an improved assay for identifying patients with RA.
- An embodiment of the invention is an assay, as in the method of the invention, where the clinical sample is used for measuring the complement activation in diseases with an inflammatory component and affecting connective tissues.
- An embodiment of the invention is an assay, as in the method of the invention, where the sample is used for measuring the complement activation in suspected RA and OA
- An embodiment of the invention is an assay, as in the method of the invention, where the sample is used for measuring the complement activation in psoriatic arthritis, chronic juvenile arthritis and pelvospondylitis.
- An embodiment of the invention is an assay, as in the method of the invention, where the sample is used for measuring the complement activation in diseases with an inflammatory components and affecting connective tissues such as RA and OA, systemic sclerosis, tendinitis and cardiovascular disease including atherosclerosis.
- An embodiment of the invention is an assay, as in the method of the invention, for monitoring the disease progression in patients with diseases with inflammatory components and affecting connective tissues such as RA and OA, systemic sclerosis, tendinitis and cardiovascular disease including atherosclerosis.
- FIG 1 shows the levels of serum COMP-C3b complexes in various disease groups. DETAILED DESCRIPTION OF THE INVENTION
- Inflammation is a component in most diseases. Previous findings indicate that molecules released from the tissue can affect complement activation as an important part in inflammation We have found that COMP is able to activate the alternative pathway of the complement system mainly through an interaction with properdin, which might direct complement attack to surfaces with exposed COMP. A pentameric structure of COMP (pCOMP) was not necessary for promoting the binding of properdin, supporting the in vivo finding that released fragments of cleaved COMP in serum have complement activating properties. Interestingly, C3 and C3b were also found to interact directly with COMP. This raises the question of whether the effect of COMP is to stabilize the alternative C3-convertase through interacting with both C3b and properdin or more simply to target complement activation by recruiting properdin and therefore providing a platform for convertase assembly.
- COMP is able to activate the alternative pathway of the complement system mainly through an interaction with properdin, which might direct complement attack to surfaces with exposed COMP.
- pCOMP pentameric structure of COMP
- C3 and C3b were
- the invention is aimed to be used to detect inflammation in general in a patient.
- the invention relates for example to a kit for determining a tissue degradation process by a method according to the invention, comprising the following components: i) A first component comprising one or more antibodies or an other ligand catching one of the components forming the COMP-C3b complex, exemplified in the present application by antibodies to COMP according to the procedure described in
- the antibodies can be ligands for COMP, antibody fragments, such as Fab or F(ab)'2 including such identified by the phage-display methods or intact antibodies binding to COMP epitopes.
- a second component comprising the detector in the form of one or more antibodies binding to the other component of the complex, exemplified in the present application by antibodies to complement factor C3b or natural breakdown products or fragments thereof.
- a third component comprising means for detecting whether one or more antibodies have reacted with one or more epitopes of the fragments as described above. This can be achieved by using a labelled detector antibody,or a labelled ligand binding to the detector antibody. In typical approaches the final detection is by commonly used approaches e.g. such including measuring activity of bound enzymes or any other probe.
- kits for determining a tissue degradation process by a method according to the invention comprising the following components:
- a first component providing an antibody for example a monoclonal antibody or an other ligand, directed against human COMP, either in solution or bound to a solid phase carrier, such as a well in a plate ,
- step ii) adding a clinical sample suspected to contain human COMP-C3b fragment complex to a first antibody of step i) and incubating the resulting mixture in aqueous solution;
- a second antibody for example a monoclonal antibody or an other ligand, directed against human C3b or fragments thereof to the mixture of step ii), said second antibody comprising a label emitting a detectable and quantifiable signal and quantifying the signal from the label, said signal being a measurement of the concentration of human COMP- C3b complex in said sample.
- the antibodies of the invention are optionally labeled with e.g. an enzyme, a radioactive, fluorescent or luminescent label in order to measure the level of bound antibody to the COMP-C3b complex present in the sample.
- the component bound to the solid phase can either be the first or the second component.
- the first component is bound to the solid phase.
- the second component comprising the antibodies of the present invention is bound to a solid phase, such as e.g. a well in a plate.
- the presence of COMP-C3b fragments in a sample can then be detected by first adding the sample to the well in the plate. If one or more complexes containing COMP-C3b fragments are present in the sample, the fragments will be bound to the solid phase via the antibodies, which bind to the COMP-C3b complex.
- the presence of fragments containing C3b epitopes can then be measured by adding the first component comprising one or more substances, which bind to one or more fragments.
- the one or more substances are optionally labelled with e.g. an enzyme or a radioactive or fluorescent label in order to measure the level of bound substance to the fragments present in the sample.
- a labeled ligand, such as an antibody, reacting with the second antibody can be used for detection.
- the invention will provide information on the synovial fluid level of complement activation induced by the process of cartilage degradation in joint disease. Serum analyses will provide information on the level of complement activation induced by the released fragments of COMP and better identify patients suffering from RA and OA. In particular information on the role of matrix components in eliciting inflammation will provide an important component in evaluating future therapeutic endavors as well as in selecting patients for such therapy.
- the invention will provide novel means to define RA patients with active disease and will include patients before current diagnosis can be made in the preclinical phase.
- the invention will define disease activity even when tissue destruction is of too low intensity to be detected by conventional current assays, including that for COMP.
- the invention will define high risk patients with a more intense disease progression.
- the invention will define patients having a higher risk of develop degenerative joint disease after trauma by analyses of synovial fluid.
- the invention will define complement activation in psoriatic arthritis, chronic juvenile arthritis and pelvospondylitis.
- the invention will identify processes in the spine leading to back pain by analyses of serum samples.
- the rationale is that COMP fragments released will activate complement to induce the inflammatory process in turn yielding pain.
- the complexes demonstrating complement activation will provide direct measures of the process.
- Blood samples are collected by venipuncture and are allowed to clot. Serum is separated by centrifugation. The samples are then diluted 1 : 10 in sample diluent (0.05 M Tris- HC1, pH 7.5, 0.90 percent (wt) NaCl, 1 percent bovine serum albumin, 0.05 percent Tween 20, 0.15 percent Kathone CG, 0.01 percent tartrazine, 0.001 M CaCl 2 , 0.01 percent bovine IgG, filtered using a 0.45 micro m filter) (12 ⁇ (microliter) sample to 108 (microliter) sample diluent).
- sample diluent 0.05 M Tris- HC1, pH 7.5, 0.90 percent (wt) NaCl, 1 percent bovine serum albumin, 0.05 percent Tween 20, 0.15 percent Kathone CG, 0.01 percent tartrazine, 0.001 M CaCl 2 , 0.01 percent bovine IgG, filtered using a 0.45 micro m filter
- Synovial fluid is collected by joint aspiration and immediately centrifuged to remove cells and any particles.
- the synovial fluid is diluted 1 : 10 in sample diluent as the serum samples.
- Example 1 As in Example 1 but also a positive pool of sera is used, representing one or more levels of complex as a direct standard to obtain quantitative measures of the COMP-C3b levels in the unknown samples.
- Cartilage matrix proteins An acidic oligomeric protein (COMP) detected only in cartilage. J Biol Chem 1992. 267: 6132-6136.
- COMP acidic oligomeric protein
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US4342566A (en) * | 1980-02-22 | 1982-08-03 | Scripps Clinic & Research Foundation | Solid phase anti-C3 assay for detection of immune complexes |
US4642284A (en) * | 1983-06-13 | 1987-02-10 | Scripps Clinic And Research Foundation | Method and system for detection of complement pathway activation |
SE452065B (en) * | 1986-04-11 | 1987-11-09 | Ulf R Nilsson | ANTIBODY PREPARATION OUR ANTIBODIES ARE SPECIFIC TO DETERMINANTS IN THE C3 B REGION OF DENATURATED HUMAN C3 AND ITS USE AND MANUFACTURING |
IL84160A0 (en) * | 1986-10-16 | 1988-03-31 | Vasocor | Method and kit for immune complex assay |
AU2028297A (en) * | 1996-03-19 | 1997-10-10 | Akzo Nobel N.V. | Method for predicting the outcome of hepatitis b infection |
SE9904236D0 (en) * | 1999-11-22 | 1999-11-22 | Ana Mar Diagnostics Ab | Immunoassay |
US20040203072A1 (en) * | 2002-11-08 | 2004-10-14 | Barnes-Jewish Hospital | Uncoupled collagen synthesis and degradation assays |
EP1766411B1 (en) * | 2004-05-24 | 2011-02-23 | AnaMar AB | Method for determining a tissue degradation process by detection of comp neoepitopes |
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JP5237366B2 (en) * | 2007-06-20 | 2013-07-17 | メルク・シャープ・アンド・ドーム・コーポレーション | Biomarkers of joint destruction for anti-IL-17A treatment for inflammatory joint diseases |
EP2307451B1 (en) * | 2008-03-21 | 2016-05-25 | Universiteit Hasselt | Biomarkers for rheumatoid arthritis |
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